1. Technical Field
The present invention relates to a process for preparing a retrovirus vector having a high titer and employed in gene therapy.
2. Prior Art
Owing to the remarkable progress in genetic engineering in recent years, there have been identified genes causative of a number of genetic diseases and thus the pathological mechanisms of these diseases have been clarified at the molecular level. Under these circumstances, studies on gene therapy have been made to transfer genes seemingly capable of ameliorating diseases into cells and some of these treatments have been already put into practical use. Also, attempts have been made to apply the gene therapy to the treatment of cancer, AIDS, etc. In the gene therapy, there are known several methods for transferring foreign genes. Among all, the most frequently employed method at the present stage is the one with the use of retrovirus vectors (Miller, A. D., Blood, 76, 271-278, 1990). Use of these vectors has the following advantages. Since the transferred gene can be surely integrated into chromosomes, it can be expressed stably over a long period of time. In addition, this method is a highly safe one with little fear of cytotoxicity. On the other hand, this method suffers from some problems such that no gene can be transferred into cells in a number of cases because of the absence of any receptor for virus envelope proteins in the target cells, that large-sized DNAs cannot be inserted thereby, and that the gene transfer thereby is available exclusively into cells which are capable of dividing. Although gene therapy with the use of retroviruses has been frequently employed, no sufficient therapeutic effect can be achieved thereby hitherto because of the above problems (Marshall, E., Science, 269, 1050-1055, 1995).
To effect the gene therapy, anyway, it is required to satisfy at least the following three conditions: 1) to efficiently transfer a desired gene into the target cells; 2) to ensure the continuous expression of the transferred gene; and 3) to be safe for the environment including the patient.
The conventional process for preparing a retrovirus vector comprises transferring a retrovirus genome containing a desired foreign gene into cells called packaging cells wherein retrovirus gag, pol and env have been expressed stably to thereby give a retrovirus containing the foreign gene in its vector DNA. However, it is difficult to prepare vectors with such high qualities as usable for clinical purposes by this process. Thus, a number of studies have been made to prevent the occurrence of a replication competent retrovirus (RCR), to produce a retrovirus having a high titer, and to elevate the titer of a retrovirus vector by improving the vector genome structure or examining the conditions for condensation or gene transfer (Vile, R. G., Gene Therapy, Churchill Livingstone, 12-30, 1995). In spite of these efforts, no technique has been established so far for a vector having a broad infection range and a high titer in a large scale stably, which is one of serious obstacles to gene therapy.
Meanwhile, studies have been energetically made for a long time by using vesicular stomatitis virus (VSV) as a model of pseudotyped viruses as the joint between retroviruses and otherviruses (Zavada, J., Arch. Virol., 50, 1, 1976). The term xe2x80x9cpseudotypexe2x80x9d means a phenomenon wherein a virus genome germinates while being surrounded by the coat protein of another virus. VSV is a virus having a negative single-stranded RNA genome and belonging to the family Rhabdovirus. It is considered that the receptors on the cell surface for the coat protein (G protein) thereof include anionic lipids such as phosphatidylserine. Thus, it is known that VSV has an extremely broad host range. It is therefore assumed that by preparing a pseudotyped retrovirus having this VSV-G gene product in the coat, genes can be efficiently transferred into cells which can be transduced at only a low or even no infective efficiency with retroviruses having the inherent envelope protein. In fact, Emi et al. (Emi, N., et al., J. Virol., 65, 1202-1207, 1991) and Yee et al. (Yee, J. K., et al., Proc. Natl. Acad. Sci. USA, 91, 9564-9568, 1994) reported a process for preparing a retrovirus vector having a VSV-G gene product as its envelope and pointed out that this pseudotyped virus enabled efficient gene transfer into cells which could have been transduced only at a low infective efficiency with a retrovirus having the inherent envelope protein.
To clinically apply such a VSV-G pseudotyped virus vector, it is necessary to establish a method for acquiring a virus with a high titer at a high reproducibility. However, it is difficult to produce the VSV-G gene product at a high level and at a high reproducibility in packaging cells, since the VSV-G gene product per se has a cytotoxicity. This is a serious problem in the development of pseudotyped vectors which are expected to be widely applicable. Recently, it was reported that VSV-G pseudotyped virus vector-producing cells can be prepared by regulating the expression of the VSV-G gene product with the use of tetracycline (Yang, Y., et al. Hum. Gene. Ther., 6, 1203-1213, 1995; Chen, S. T., et al., Prc. Natl. Acad. Sci. USA, 93, 10057-10062, 1996; and Ory, D. S., et al., Proc. Natl. Acad. Sci. USA, 93, 11400-11406, 1996). However, there still remain some problems in these reports such that the regulation of the expression of the VSV-G gene product was not completely regulated by tetracycline and, therefore, the producing cells might be still re-infected with about 102 to 104 i.u./ml of the VSV-G pseudotyped virus vector thus produced; and that the stability of the packaging cells over a long period of time was still unreliable, since they were made of the co-transfection of a DNA with the VSV-G expression and another DNA with the drug-resistance gene expression.
It is also known that, when a foreign gene to be transferred into target cells with the use of a retrovirus strongly affects the cells, the virus carrying this foreign gene in its virus vector DNA cannot be recovered stably, since the foreign gene product affects in the virus-producing cells (i.e., the packaging cells containing and expressing the vector DNA) per se (Pear, W. S. et al., Proc. Natl. Acad. Sci. USA, 90, 8392-8396, 1993).
In the present description, the term xe2x80x9cpseudotyped virus vectorxe2x80x9d refers to a retrovirus vector having a VSV-G gene product in its envelope, while the term xe2x80x9cretrovirus vectorxe2x80x9d refers to both a retrovirus vector having the inherent envelope protein and a xe2x80x9cpseudotyped virus vectorxe2x80x9d.
The term xe2x80x9cprepackaging cellsxe2x80x9d refers to cells in which gag and pol of a retrovirus can be expressed and env thereof cannot be expressed as usual before recombinase was introduced to cells. The term xe2x80x9cprepackaging cells containing avector DNAxe2x80x9d refers to the prepackaging cells as defined above into which a vector DNA has been transferred. Further, the term xe2x80x9cpackaging cells containing a vector DNAxe2x80x9d refers to cells capable of producing a virus when a recombinase is transferred thereinto.
Moreover, the term xe2x80x9cdrug resistance genexe2x80x9d as used herein refers to all of low-efficient drug resistance genes, short-lived transcript drug resistance genes having a base sequence of a short-lived mRNA of a drug resistance gene and conventional drug resistance genes.
Under these circumstances, an object of the present invention is to establish a process whereby a retrovirus vector for specifically transferring foreign genes including those affecting cells into target cells over a broad range and expressing the genes therein can be stably produced at a high titer by strictly regulating, compared with the conventional processes, cytotoxic virus structural proteins and vector DNA with a cytotoxic or cell-affecting protein; to establish a process for elevating the recovery yield of a retrovirus vector by inhibiting the reinfection of producing cells with a pseudotyped retrovirus vector; and to efficiently screen high-expression cell clones by using a low-efficient drug resistance gene or a short-lived transcript drug resistance gene in the transcription of two genes by a recombinase with the use of the same promoter.
To solve the above problems, the present inventors have conducted intensive studies.
A DNA wherein an loxP sequence, a drug resistance gene, a polyA addition signal, an loxP sequence, a VSV-G gene and a polyA addition signal are arranged in this order downstream of a potent promoter is transferred into cells having gag and pol genes of a retrovirus transferred thereinto. Then the resultant cells are screened with the use of the drug to prepare prepackaging cells. The prepackaging cells thus prepared are transduced with a retrovirus vector containing a desired gene inserted in its vector DNA to thereby transfer the gene into the prepackaging cells. At the same time, a recombinase is introduced to the cell. Thus, the VSV-G gene product can be expressed at a high level within a short period of time with the use of the same potent promoter as the one employed for expressing the drug resistance gene. As a result, a pseudotyped virus vector having a high titer can be successfully prepared in a large amount prior to the appearance of the cytotoxicity of both the VSV-G gene product and foreign gene product in vector DNA. The present invention has been thus completed.
The present inventors have also found that the producing cells are reinfected with the pseudotyped retrovirus vector and the reinfection can be inhibited by adding a negatively charged, high-molecular weight substance to the liquid culture medium, thus enhancing the recovery yield.
They have also utilized the phenomenon that, in the preparation of the above-mentioned prepackaging cells, cells requiring the expression of a stronger resistance marker can be efficiently screened by using as a drug resistance marker gene one the function of which has been deteriorated by substitution, insertion or deletion in the base sequence in the coding region, one the translation efficiency of which has been lowered by substitution, insertion or deletion in the base sequence in the untranslated region (i.e., a low-efficient drug resistance gene), or one the stability of the mRNA produced by which has been lowered (i.e., a short-lived transcript drug resistance gene). In the present invention, use is made of these short-lived transcript drug resistance genes thus devised. Furthermore, it has become possible to establish the enhanced expression of the VSV-G gene product by transferring into the cells a retrovirus vector having the desired gene or its DNA inserted thereinto and treating with a recombinase. Thus, the present inventors have succeeded in the production of a large amount of a pseudotyped vector having a high titer.
The present invention relates to processes for preparing retrovirus vectors for gene therapy which comprise transferring a DNA construction (hereinafter referred to as a DNA construction (A)) for regulating the expression of a virus structural protein by using a recombinase and its recognition sequence and another DNA construction (hereinafter referred to as a DNA construction (B)) for regulating the expression of a foreign gene encoded in a vector DNA, by using a recombinase and its recognition sequence into retrovirus gag-pol-producing cells followed by the transfer of a DNA with the recombinase expression thereinto. More particularly, it relates to: 1) a process for preparing a retrovirus vector for gene therapy which comprises transferring a DNA construction (A) wherein a promoter, a recombinase recognition sequence, a drug resistance gene, a polyA addition signal, a recombinase recognition sequence, a virus structural protein gene and a polyA addition signal are arranged in this order and another DNA construction (B) wherein the LTR of a retrovirus genome and a packaging signal are followed by a recombinase recognition sequence, a drug resistance gene, a polyA addition signal, a recombinase recognition sequence, a foreign gene and LTR arranged in this order into retrovirus gag-pol-producing cells followed by the transfer of a DNA with the recombinase expression thereinto; 2) a process for preparing a retrovirus vector for gene therapy which comprises transferring into retrovirus gag-pol-env-producing cells a DNA construction (B) wherein the LTR of a retrovirus genome and a packaging signal are followed by a recombinase recognition sequence, a drug resistance gene, a polyA addition signal, a recombinase recognition sequence, a foreign gene and LTR arranged in this order followed by the transfer of a DNA with the recombinase expression thereinto; 3) a process for preparing a retrovirus vector for gene therapy which comprises transferring a DNA construction (A) wherein a promoter, a recombinase recognition sequence, a drug resistance gene, a polyA addition signal, a recombinase recognition sequence, a virus structural protein gene and a polyA addition signal are arranged in this order and a retrovirus vector DNA encoding a foreign gene into retrovirus gag-pol-producing cells followed by the transfer of a DNA with the recombinase expression thereinto; 4) a DNA construction (A) wherein a promoter, a recombinase recognition sequence, a drug resistance gene, a polyA addition signal, a recombinase recognition sequence, a virus structural protein gene and a polyA addition signal are arranged in this order; 5) a DNA construction (B) wherein the LTR of a retrovirus genome and a packaging signal are followed by a recombinase recognition sequence, a drug resistance gene, a polyA addition signal, a recombinase recognition sequence, a foreign gene and LTR arranged in this order; 6) a DNA construction (A) wherein the promoter is CAG; 7) a DNA construction (A) or a DNA construction (B) wherein the recombinase and its recognition sequence are Cre recombinase and loxP sequence respectively; 8) a DNA construction (A) or a DNA construction (B) wherein the drug resistance gene is a neomycin-resistance gene, a puromycin-resistance gene or a hygromycin-resistance gene; 9) a DNA construction (A) or a DNA construction (B) wherein the drug resistance gene is a low-efficient drug resistance gene or a short-lived transcript drug resistance gene having a short-lived mRNA base sequence; 10) a DNA construction (A) or a DNA construction (B) wherein the low-efficient drug resistance gene or the short-lived transcript drug resistance gene is one originating in a neomycin resistance gene, a puromycin resistance gene or a hygromycin resistance gene; 11) a short-lived transcript drug resistance gene characterized by having a short-lived mRNA base sequence of a neomycin resistance gene, a puromycin resistance gene or a hygromycin resistance gene; 12) a short-lived transcript drug resistance gene wherein the mRNA has been made short-lived with an mRNA-unstabilizing signal originating in c-fos; 13) a DNA construction (A) or a DNA construction (B) wherein the polyA addition signal is one originating in SV40 or xcex2-globin; 14) a DNA construction (A) wherein the virus structural protein gene is a DNA encoding vesicular stomatitis virus (VSV) G protein (VSV-G); 15) the DNA construction (B) as set forth in claim 2, wherein the retrovirus genome is one originating in Moloney murine leukemia virus (MoMLV); 16) a DNA construction (B) wherein the retrovirus genome is one originating in a lentivirus; 17) a DNA construction (B) wherein the foreign gene is a gene aiming at cell transfer for gene therapy; 18) a DNA construction (B) wherein the foreign gene is a gene of a cytotoxic protein; 19) a DNA construction (A) wherein a CAG promoter, an loxP sequence, a drug resistance gene, a polyA addition signal, an loxP sequence, a VSV-G gene and a polyA addition signal are arranged in this order; 20) a DNA construction (B) wherein the LTR of a retrovirus genome and a packaging signal are followed by an loxP sequence, a drug resistance gene, a polyA addition signal, an loxP sequence, a foreign gene and LTR arranged in this order; 21) a prepackaging cell for producing a retrovirus vector wherein a DNA construction (A) has been transferred into a retrovirus gag-pol-producing cell; 22) a virus vector DNA-containing prepackaging cell for producing a retrovirus vector wherein a DNA construction (B) has been transferred into a retrovirus gag-pol-env-producing cell; 23) a virus vector DNA-containing prepackaging cell for producing a retrovirus vector wherein a DNA construction (A) and a DNA construction (B) have been transferred into a retrovirus gag-pol-producing cell; 24) a virus vector DNA-containing prepackaging cell for producing a retrovirus vector wherein a DNA construction (A) and a virus vector DNA encoding a foreign gene have been transferred into a retrovirus gag-pol-producing cell; 25) a prepackaging cell for producing a retrovirus vector wherein the retrovirus is murine leukemia virus (MLV); 26) a process for preparing a pseudotyped retrovirus wherein a negatively charged, high-molecular weight substance is contained in the liquid culture medium; and 27) a process for producing a pseudotyped retrovirus wherein the negatively charged, high-molecular weight substance is one selected from among heparin, heparan sulfate and chondroitin sulfate.